U.S. patent application number 16/218574 was filed with the patent office on 2019-06-13 for genetically modified tobacco.
The applicant listed for this patent is MyMD Pharmaceuticals, Inc.. Invention is credited to Jonnie R. Williams.
Application Number | 20190177737 16/218574 |
Document ID | / |
Family ID | 66629324 |
Filed Date | 2019-06-13 |
United States Patent
Application |
20190177737 |
Kind Code |
A1 |
Williams; Jonnie R. |
June 13, 2019 |
Genetically Modified Tobacco
Abstract
A method of increasing the level of isomyosmine in a tobacco
plant that includes genetically modifying the tobacco plant to
induce the tobacco plant to produce a tobacco with increased levels
of isomyosmine in the tobacco. Further, the tobacco may have
decreased levels of nicotine. In some examples, the decreased
levels of nicotine are the result of a second genetic modification
to the tobacco plant.
Inventors: |
Williams; Jonnie R.;
(Sarasota, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MyMD Pharmaceuticals, Inc. |
Tampa |
FL |
US |
|
|
Family ID: |
66629324 |
Appl. No.: |
16/218574 |
Filed: |
December 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62598052 |
Dec 13, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01H 5/12 20130101; C12N
15/8243 20130101; A01H 6/823 20180501 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01H 6/82 20060101 A01H006/82 |
Claims
1. A method of increasing a level of isomyosmine in a tobacco plant
comprising genetically modifying a tobacco plant with a genetic
modification, wherein the genetic modification induces the tobacco
plant to produce a tobacco with increased levels of isomyosmine in
the tobacco; and wherein the tobacco has decreased levels of
nicotine.
2. The method of claim 1, wherein the genetically modified tobacco
plant has a nicotine converter rate of about 3.0% or greater.
3. The method of claim 1, wherein the tobacco has a nicotine
content of less than about 2 mg/g.
4. The method of claim 1, wherein the tobacco has an isomyosmine
content of at least 0.1 mg/g.
5. The method of claim 1, wherein the tobacco has an isomyosmine
content from about 0.001 mg/g to about 10 mg/g.
6. The method of claim 1, wherein the tobacco plant is a solanecea
plant.
7. The method of claim 1, wherein the tobacco has decreased levels
of nicotine due to a second genetic modification to the tobacco
plant.
8. The method of claim 7, wherein the second genetic modification
to the tobacco plant inhibits the expression of a
methylenetetrahydrofolate reductase (MTHFR) gene.
9. The method of claim 8, wherein the inhibition of the expression
of the MTHFR gene induces an increased expression of a nicotine
N-demethylase (CYP82E4) gene.
10. The method of claim 9, wherein the increased expression of the
CYP82E4 gene results in an increased nicotine-to-nornicotine
conversion rate, and wherein the increased nicotine-to-nornicotine
conversion rate decreases the levels of nicotine in the
tobacco.
11. A tobacco comprising an increased level of isomyosmine, and a
reduced level of nicotine.
12. A tobacco product comprising the tobacco of claim 11.
13. The tobacco product of claim 12, wherein the tobacco product is
a cigarette, cigar, pipe tobacco, smokeless tobacco, chewing
tobacco, capsule, tablet, or lozenge.
14. The tobacco of claim 11, wherein the tobacco is produced from a
genetically modified plant, wherein the tobacco plant has a genetic
modification causing a nicotine converter rate of about 3.0% or
more.
15. The tobacco of claim 11, wherein the tobacco is processed, and
wherein the processing reduces a nicotine content of the
tobacco.
16. The tobacco of claim 11, wherein the tobacco is produced from a
genetically modified tobacco plant, wherein the tobacco plant has a
genetic modification causing an increased level of isomyosmine in
the tobacco.
17. The tobacco of claim 11, wherein the isomyosmine is
synthetic.
18. The tobacco of claim 11, wherein the tobacco contains an
isomyosmine content of at least 0.1 mg/g.
19. The tobacco of claim 11, wherein the nicotine content is less
than about 2 mg/g.
20. A genetically modified tobacco plant comprising a genetic
modification, wherein the genetic modification induces a tobacco
plant to produce a tobacco with increased levels of isomyosmine;
and wherein the tobacco has decreased levels of nicotine.
21. The genetically modified tobacco plant of claim 20, wherein the
tobacco plant has a second genetic modification, wherein the second
genetic modification induces the tobacco plant to produce the
tobacco with decreased levels of nicotine.
22. The genetically modified tobacco plant of claim 21, wherein the
second genetic modification to the tobacco plant inhibits the
expression of a MTHFR gene, wherein the inhibition of the
expression of the MTHFR gene induces an increased expression of a
CYP82E4 gene, wherein the increased expression of the CYP82E4 gene
results in an increased nicotine-to-nornicotine conversion rate,
and wherein the increased nicotine-to-nornicotine conversion rate
decreases the levels of nicotine in the tobacco.
23. The genetically modified tobacco plant of claim 20, wherein the
genetically modified tobacco plant has a nicotine converter rate of
about 3.0% or greater.
24. The genetically modified tobacco plant of claim 20, wherein the
tobacco has a nicotine content of less than about 2 mg/g.
25. The genetically modified tobacco plant of claim 20, wherein the
tobacco has an isomyosmine content of at least 0.1 mg/g.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority under 35 U.S.C. .sctn.
119(e) to U.S. Application No. 62/598,052, filed Dec. 13, 2017, the
entire contents of which are incorporated herein by reference.
BACKGROUND
[0002] Nicotine is the primary basis for tobacco addiction and the
tobacco industry has experienced a renewed interest in producing
tobacco plants and tobacco products with reduced levels of
nicotine. Efforts to reduce the level of nicotine content in
tobacco have existed for much of the history of tobacco
cultivation. Techniques such as plant breeding, as well as chemical
and processing methods have been used to reduce nicotine levels in
tobacco. Modern efforts include genetically modified tobacco plants
that produce reduced levels of nicotine. An individual's desire,
however, to achieve the same physiological effects experienced from
smoking or using a tobacco product with conventional levels of
nicotine have inhibited the success of reduced nicotine
products.
[0003] It would be desirable to reduce the levels of nicotine in
tobacco products while still providing an individual with the
pleasurable effects associated with tobacco use. Described herein
are tobacco products featuring reduced levels of nicotine and
increased levels of isomyosmine. Increased levels of isomyosmine in
tobacco products may be achieved by the genetic engineering of
tobacco plants or by introducing isomyosmine into tobacco material
during processing.
[0004] A reduced nicotine tobacco product that has an increased
content of isomyosmine may provide an attractive alternative to
conventional tobacco products and may provide a viable option for
reducing or eliminating cravings for nicotine or dependence on
nicotine.
SUMMARY
[0005] In accordance with aspects disclosed herein, methods of
reducing the levels of nicotine and increasing the levels of
isomyosmine in a genetically modified tobacco plant are disclosed.
In other aspects, methods of increasing the levels of isomyosmine
in a tobacco plant by genetically modifying the tobacco plant in
such a manner that induces the tobacco plant to produce tobacco
with increased levels of the alkaloid isomyosmine, and the tobacco
may have decreased levels of nicotine. In other aspects, the
tobacco plant may also produce tobacco with decreased levels of
nicotine. In other examples, the tobacco plant may include a first
genetic modification that induces the plant to produce increased
levels of isomyosmine in the tobacco, and a second genetic
modification that induces the tobacco plant to produce tobacco with
decreased levels of nicotine content.
[0006] In accordance with another aspect, a method of increasing
the isomyosmine in a tobacco plant that is genetically modified is
disclosed in which the genetically modified plant has a nicotine
converter rate of at least 3% or greater. In other aspects, the
tobacco produced by the disclosed method has a nicotine content of
less than about 2 mg/g. In still other aspects, the disclosed
method includes a tobacco with an isomyosmine content from about
0.001 mg/g to about 10 mg/g, from about 0.01 mg/g to about 10 mg/g,
or from about 0.1 mg/g to about 100 mg/g.
[0007] In accordance with another aspect, a method of increasing
the isomyosmine in a tobacco plant that is genetically modified is
disclosed in which the genetically modified plant is a solanecea
plant. In yet another aspect, the genetic modification to the
tobacco plant induces an increased production of isomyosmine, and
the increased production of the isomyosmine results in the
increased levels of isomyosmine in the tobacco. In still other
aspects, the genetically modified tobacco plant includes a second
genetic modification that decreases the levels of nicotine in the
tobacco. In another aspect, the second genetic modification
inhibits the expression of the methylenetetrahydrofolate reductase
(MTHFR) gene of the tobacco plant and the inhibition of the
expression of the MTHFR gene induces an increased expression of the
nicotine N-demethylase (CYP82E4) gene. The increased expression or
overexpression of the CYP82E4 gene results in an increased
nicotine-to-nornicotine conversion rate, and the increased
nicotine-to-nornicotine conversion rate decreases the levels of
nicotine in the tobacco.
[0008] In accordance with other aspects herein, compositions of
tobacco with increased levels of isomyosmine and a reduced level of
nicotine are disclosed. In other examples, the tobacco is produced
from a genetically modified tobacco plant that induces the
production of increased levels of isomyosmine. In still other
aspects, the isomyosmine is synthetically added to the tobacco. In
still other aspects, the composition of tobacco has decreased
levels of nicotine as a result of a genetic modification that
induces a tobacco plant to produce tobacco with lower levels of
nicotine. In still other examples, the composition of tobacco is
chemically treated or processed to reduce the level of
nicotine.
[0009] In accordance with certain aspects disclosed herein, the
composition of tobacco may be formed into a tobacco product such as
a cigarette, cigar, pipe tobacco, smokeless tobacco, chewing
tobacco, capsule, tablet, or lozenge.
[0010] In accordance with other aspects, compositions of tobacco
with increased levels of isomyosmine, and reduced levels of
nicotine are disclosed in which the tobacco is produced from a
genetically modified plant, and the tobacco plant has a genetic
modification that causes a nicotine converter rate of about 3.0% or
more, or at least 3.0%. In another aspect, the tobacco is
chemically treated or processed to reduce the nicotine content. In
still another aspect, the tobacco is produced from a genetically
modified tobacco plant in which the tobacco plant has a genetic
modification that causes an increased level of isomyosmine in the
tobacco. In other aspects, the isomyosmine found in the tobacco is
synthetic. In still other aspects, the tobacco has an isomyosmine
content from about 0.001 mg/g to about 10 mg/g, from about 0.01
mg/g to about 10 mg/g, or from about 0.1 mg/g to about 100 mg/g. In
still other aspects, the tobacco has a nicotine content of less
than about 2 mg/g.
[0011] In accordance with another aspect, a tobacco plant is
disclosed that produces tobacco with increased levels of
isomyosmine due to a genetic modification in the tobacco plant. In
accordance with another aspect, the tobacco plant also produces
tobacco with reduced levels of nicotine. In other aspects, the
reduced levels of nicotine in the tobacco produced by the tobacco
plant are the result of a genetic modification that causes the
plant to produce lower levels of nicotine in the tobacco. In yet
other aspects, the tobacco is chemically treated or processed to
reduce the levels of nicotine in the tobacco. In certain aspects,
the genetically modified tobacco plant has a first genetic
modification that induces the plant to produce tobacco with
increased levels of isomyosmine, and a second genetic modification
that inhibits the expression of the MTHFR gene. The inhibition of
the expression of the MTHFR gene induces an increased expression of
the CYP82E4 gene that results in an increased
nicotine-to-nornicotine conversion rate. The increased
nicotine-to-nornicotine conversion rate decreases the levels of
nicotine in the tobacco.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] A more complete understanding of the present invention and
certain advantages thereof may be acquired by referring to the
following detailed description in consideration with the
accompanying drawings, in which:
[0013] FIG. 1 is a schematic representing the conversion of
nicotine to nornicotine mediated by nicotine N-demethylase gene
CYP82E4.
[0014] FIG. 2 is a schematic representing the molecular structures
of several tobacco alkaloids.
DETAILED DESCRIPTION
[0015] In tobacco plants, nicotine and other alkaloids are
synthesized in the roots. The nicotine and other alkaloids are
transported by the xylem to the leaf. Typically, 90% to 95% of the
alkaloid content in commercial tobacco plants is nicotine, which is
about 2% to 5% of dry leaf weight. Saitoh F, Nona M, Kawashima N
(1985), The alkaloid contents of sixty Nicotiana species,
Phytochemistry 24: 477-480. As depicted in FIG. 1, nicotine may
undergo N-demethylation in the leaf to produce the alkaloid
nornicotine. The biosynthesis of nornicotine from nicotine is
largely mediated by the nicotine N-demethylase gene CYP82E4.
Tobacco plants with a nicotine-to-nornicotine conversion rate (NCR)
greater than about 3% have been defined as "convertors" and were
previously held as commercially undesirable due to the reduced
nicotine levels produced by the tobacco plant. Jack A, Fannin N,
Bush L P (2007), Implications of reducing nornicotine accumulation
in burley tobacco, Appendix A: the LC protocol. Rec Adv Tob Sci 33:
58-79.
[0016] According to another aspect, the tobacco disclosed herein
has a nicotine content of, for example, at least, greater than,
less than, equal to, or any number in between about 0.001 mg/g,
0.01 mg/g, 0.10 mg/g, 0.15 mg/g, 0.20 mg/g, 0.25 mg/g, 0.30 mg/g,
0.35 mg/g, 0.40 mg/g, 0.45 mg/g, 0.50 mg/g, 0.55 mg/g, 0.60 mg/g,
0.65 mg/g, 0.70 mg/g, 0.75 mg/g, 0.80 mg/g, 0.85 mg/g, 0.90 mg/g,
0.95 mg/g, 1.00 mg/g, 1.10 mg/g, 1.15 mg/g, 1.20 mg/g, 1.25 mg/g,
1.30 mg/g, 1.35 mg/g, 1.40 mg/g, 1.45 mg/g, 1.50 mg/g, 1.55 mg/g,
1.60 mg/g, 1.65 mg/g, 1.70 mg/g, 1.75 mg/g, 1.80 mg/g, 1.85 mg/g,
1.90 mg/g, 1.95 mg/g, 2.00 mg/g, 2.10 mg/g, 2.15 mg/g, 2.20 mg/g,
2.25 mg/g, 2.30 mg/g, 2.35 mg/g, 2.40 mg/g, 2.45 mg/g, 2.50 mg/g,
2.55 mg/g, 2.60 mg/g, 2.65 mg/g, 2.70 mg/g, 2.75 mg/g, 2.80 mg/g,
2.85 mg/g, 2.90 mg/g, 2.95 mg/g, 3.00 mg/g, 3.10 mg/g, 3.15 mg/g,
3.20 mg/g, 3.25 mg/g, 3.30 mg/g, 3.35 mg/g, 3.40 mg/g, 3.45 mg/g,
3.50 mg/g, 3.55 mg/g, 3.60 mg/g, 3.65 mg/g, 3.70 mg/g, 3.75 mg/g,
3.80 mg/g, 3.85 mg/g, 3.90 mg/g, 3.95 mg/g, 4.00 mg/g, 4.10 mg/g,
4.15 mg/g, 4.20 mg/g, 4.25 mg/g, 4.30 mg/g, 4.35 mg/g, 4.40 mg/g,
4.45 mg/g, 4.40 mg/g, 4.45 mg/g, 4.50 mg/g, 4.55 mg/g, 4.60 mg/g,
4.65 mg/g, 4.70 mg/g, 4.75 mg/g, 4.80 mg/g, 4.85 mg/g, 4.90 mg/g,
4.95 mg/g, 5.00 mg/g, 5.50 mg/g, 5.70 mg/g, 6.00 mg/g, 6.50 mg/g
mg/g, 6.70 mg/g, 7.00 mg/g, 7.50 mg/g, 7.70 mg/g, 8.00 mg/g, 8.50
mg/g, 8.70 mg/g, 9.00 mg/g, 9.50 mg/g, 9.70 mg/g, 10.0 mg/g, 10.5
mg/g, 10.7 mg/g, and 11.0 mg/g, and at least, greater than, less
than, equal to, or any number in between about 0.001 .mu.g/g, 0.002
.mu.g/g, 0.003 .mu.g/g, 0.004 .mu.g/g, 0.005 .mu.g/g, 0.006
.mu.g/g, 0.007 .mu.g/g, 0.008 .mu.g/g, 0.009 .mu.g/g, 0.01 .mu.g/g,
0.02 .mu.g/g, 0.03 .mu.g/g, 0.04 .mu.g/g, 0.05 .mu.g/g, 0.06
.mu.g/g, 0.07 .mu.g/g, 0.08 .mu.g/g, 0.09 .mu.g/g, 0.01 .mu.g/g,
0.15 .mu.g/g, 0.20 .mu.g/g, 0.25 .mu.g/g, 0.30 .mu.g/g, 0.35
.mu.g/g, 0.40 .mu.g/g, 0.45 .mu.g/g, 0.5 .mu.g/g, 0.55 .mu.g/g,
0.60 .mu.g/g, 0.65 .mu.g/g, 0.70 .mu.g/g, 0.75 .mu.g/g, 0.80
.mu.g/g, 0.85 .mu.g/g, 0.90 .mu.g/g, 0.95 .mu.g/g, 1.00 .mu.g/g,
1.10 .mu.g/g, 1.15 .mu.g/g, 1.20 .mu.g/g, 1.25 .mu.g/g, 1.30
.mu.g/g, 1.35 .mu.g/g, 1.40 .mu.g/g, 1.45 .mu.g/g, 1.50 .mu.g/g,
1.55 .mu.g/g, 1.60 .mu.g/g, 1.65 .mu.g/g, 1.70 .mu.g/g, 1.75
.mu.g/g, 1.80 .mu.g/g, 1.85 .mu.g/g, 1.90 .mu.g/g, 1.95 .mu.g/g,
2.00 .mu.g/g, 2.10 .mu.g/g, 2.15 .mu.g/g, 2.20 .mu.g/g, 2.25
.mu.g/g, 2.30 .mu.g/g, 2.35 .mu.g/g, 2.40 .mu.g/g, 2.45 .mu.g/g,
and 2.50 .mu.g/g.
[0017] Techniques for alteration of tobacco to reduce nicotine
levels are known in the art and described, for example, in U.S.
Patent Publication 2010/0206317, U.S. Pat. Nos. 3,901,248 and
6,907,887, the disclosures of which are hereby incorporated by
reference in their entirety. Tobacco may be chemically treated to
remove nicotine or a tobacco plant may be selectively bred to
produce low alkaloid or low nicotine levels, such as tobacco plants
having one or more mutations in genes encoding enzymes or proteins
involved in nicotine biosynthesis. Other methods such as microbial
enzymatic degradation, chemical extraction, or high pressure
extraction may be employed to reduce nicotine levels in tobacco
plants. More recently, techniques in genetic engineering and
chemically induced gene suppression or expression have been used to
reduce the nicotine levels in tobacco plants. Any one or more of
these techniques can be used to create a tobacco or tobacco product
used with the disclosures herein.
[0018] According to another aspect, a tobacco plant may be
genetically modified to express, repress, alter, or mutate a gene
or genes involved in nicotine and/or other alkaloid biosynthesis.
According to yet another aspect, a genetically modified tobacco
plant disclosed herein may produce low levels of nicotine and
elevated levels of isomyosmine due to a modified gene or genes
encoding an enzyme(s) or protein(s) involved in nicotine and/or
isomyosmine biosynthesis.
[0019] A genetic engineering technique to decrease the nicotine
content in a tobacco plant involves manipulating the
methylenetetrahydrofolate reductase (MTHFR) gene in a tobacco
plant. See Chiu-Yueh Hung, et al. (2013), Alteration of the
Alkaloid Profile in Genetically Modified Tobacco Reveals a Role of
Methylenetetrahydrofolate Reductase in Nicotine N-Demethylation,
Plant Physiology February 2013, 161 (2) 1049-1060; DOI: 10.1104/pp.
112.209247. It is known that an altered MTHFR gene expression
negatively regulates the expression of the nicotine N-demethylase
gene CYP82E4. A genetically modified tobacco plant that suppresses
the MTHFR gene induces the expression of the nicotine N-demethylase
gene CYP82E4. Thus, a transgenic plan that suppresses the MTHFR
gene favors nicotine N-demethylation in tobacco leaves and results
in lower levels of nicotine production.
[0020] As shown in FIG. 2, a number of structurally related
alkaloids are found in tobacco other than nicotine, including
nicotine, nornicotine, myosmine, anabasine, anatabine,
isonicoteine, and isomyosmine. Isomyosmine
(3-(3,4-dihydro-2H-pyrrol-2-yl)-pyridine), shown below, is a
nicotine related alkaloid present in solanecea plants containing
nicotine.
##STR00001##
[0021] In the examples in which isomyosmine is added to tobacco
material, isomyosmine may be prepared synthetically using known
techniques or obtained from chemical suppliers. As an alternative
to synthetic preparation, isomyosmine may be obtained by extraction
from tobacco or other materials in which it occurs naturally. For
example, tobacco material may extracted with a solvent, such as
water, ethanol, steam, and/or carbon dioxide. The resulting
solution contains the soluble components of the tobacco, including
alkaloids such as nicotine, isomyosmine, and myosmine. Isomyosmine
may be purified using known techniques such as liquid
chromatography. The purified isomyosmine may then be added to
tobacco compositions or products.
[0022] According to another aspect, the tobacco disclosed herein
has an isomyosmine content of, for example, at least, greater than,
less than, equal to, or any number in between about 0.001 mg/g,
0.01 mg/g, 0.10 mg/g, 0.15 mg/g, 0.20 mg/g, 0.25 mg/g, 0.30 mg/g,
0.35 mg/g, 0.40 mg/g, 0.45 mg/g, 0.50 mg/g, 0.55 mg/g, 0.60 mg/g,
0.65 mg/g, 0.70 mg/g, 0.75 mg/g, 0.80 mg/g, 0.85 mg/g, 0.90 mg/g,
0.95 mg/g, 1.00 mg/g, 1.10 mg/g, 1.15 mg/g, 1.20 mg/g, 1.25 mg/g,
1.30 mg/g, 1.35 mg/g, 1.40 mg/g, 1.45 mg/g, 1.50 mg/g, 1.55 mg/g,
1.60 mg/g, 1.65 mg/g, 1.70 mg/g, 1.75 mg/g, 1.80 mg/g, 1.85 mg/g,
1.90 mg/g, 1.95 mg/g, 2.00 mg/g, 2.10 mg/g, 2.15 mg/g, 2.20 mg/g,
2.25 mg/g, 2.30 mg/g, 2.35 mg/g, 2.40 mg/g, 2.45 mg/g, 2.50 mg/g,
2.55 mg/g, 2.60 mg/g, 2.65 mg/g, 2.70 mg/g, 2.75 mg/g, 2.80 mg/g,
2.85 mg/g, 2.90 mg/g, 2.95 mg/g, 3.00 mg/g, 3.10 mg/g, 3.15 mg/g,
3.20 mg/g, 3.25 mg/g, 3.30 mg/g, 3.35 mg/g, 3.40 mg/g, 3.45 mg/g,
3.50 mg/g, 3.55 mg/g, 3.60 mg/g, 3.65 mg/g, 3.70 mg/g, 3.75 mg/g,
3.80 mg/g, 3.85 mg/g, 3.90 mg/g, 3.95 mg/g, 4.00 mg/g, 4.10 mg/g,
4.15 mg/g, 4.20 mg/g, 4.25 mg/g, 4.30 mg/g, 4.35 mg/g, 4.40 mg/g,
4.45 mg/g, 4.40 mg/g, 4.45 mg/g, 4.50 mg/g, 4.55 mg/g, 4.60 mg/g,
4.65 mg/g, 4.70 mg/g, 4.75 mg/g, 4.80 mg/g, 4.85 mg/g, 4.90 mg/g,
4.95 mg/g, 5.00 mg/g, 5.50 mg/g, 5.70 mg/g, 6.00 mg/g, 6.50 mg/g
mg/g, 6.70 mg/g, 7.00 mg/g, 7.50 mg/g, 7.70 mg/g, 8.00 mg/g, 8.50
mg/g, 8.70 mg/g, 9.00 mg/g, 9.50 mg/g, 9.70 mg/g, 10.0 mg/g, 10.5
mg/g, 10.7 mg/g, and 11.0 mg/g.
[0023] The tobacco plant disclosed herein includes any plant of the
genus Nicotiana of the nightshade family (Solanaceae). Another
alternative to naturally produced tobacco related products, and/or
tobacco plants with synthetic or naturally produced isomyosmine
content is to engineer a transgenic solanecea plant that produces
tobacco with elevated or increased levels of isomyosmine. It is
known that tobacco plant metabolisms are controlled by genetic as
well as environmental factors. A solanecea plant may be genetically
modifying to increase the levels of isomyosmine in the tobacco
leaves used in tobacco compositions. A solanecea plant may also be
genetically modifying to decrease the levels of nicotine in the
tobacco leaves used in tobacco compositions. A transgenic solanecea
plant can be engineered to produce low levels of nicotine and/or
elevated levels of isomyosmine due to a modified gene or genes
encoding an enzyme(s) or protein(s) involved in nicotine and/or
isomyosmine biosynthesis. According to another aspect, a solanecea
plant may be genetically modified to express, repress, alter, or
mutate a gene or genes involved in isomyosmine biosynthesis.
[0024] As used herein, the terms "protein" and "polypeptide" and
"peptide" are used interchangeably herein to designate a series of
amino acid residues, connected to each other by peptide bonds
between the alpha-amino and carboxy groups of adjacent residues.
The terms "protein," "peptide," and "polypeptide" refer to a
polymer of amino acids, including modified amino acids (e.g.,
phosphorylated, glycated, glycosylated, etc.) and amino acid
analogs, regardless of its size or function. "Protein" and
"polypeptide" are often used in reference to relatively large
polypeptides, whereas the term "peptide" is often used in reference
to small polypeptides, but usage of these terms in the art
overlaps. The terms "protein" and "peptide" are used
interchangeably herein when referring to a gene product and
fragments thereof. Thus, exemplary polypeptides, peptides, or
proteins include gene products, naturally occurring proteins,
homologs, orthologs, paralogs, fragments and other equivalents,
variants, fragments, and analogs of the foregoing.
[0025] According to one aspect, genes encoding a polypeptide
related to isomyosmine biosynthesis may be utilized to overexpress
or inhibit the expression of the polypeptide in a tobacco plant in
which the polypeptide may normally be found. In other aspects, the
gene may be used to design a polynucleotide that inhibits or
induces the expression of the gene, and the polynucleotide may be
introduced into a cell of the tobacco plant.
[0026] An "amino acid sequence" may be determined directly for a
protein or peptide, or inferred from the corresponding nucleic acid
sequence. A "nucleic acid" or "nucleic acid sequence" may be any
molecule, preferably a polymeric molecule, incorporating units of
ribonucleic acid, deoxyribonucleic acid or an analog thereof. The
nucleic acid can be either single-stranded or double-stranded. A
single-stranded nucleic acid can be one nucleic acid strand of a
denatured double-stranded DNA. Alternatively, it can be a
single-stranded nucleic acid not derived from any double-stranded
DNA. In one aspect, the nucleic acid can be DNA. In another aspect,
the nucleic acid can be RNA. Suitable nucleic acid molecules are
DNA, including genomic DNA or cDNA. Other suitable nucleic acid
molecules are RNA, including mRNA. "Heterologous" as applied to
nucleic acids is of different origin than that of the natural
tobacco plant cell.
[0027] A "vector" refers to a piece of DNA, either single or double
stranded. The vector can be for example, of plasmid or viral
origin, which typically encodes a selectable or screenable marker
or transgenes. The vector is used to transport the foreign or
heterologous DNA into a suitable host cell. Once in the host cell,
the vector can replicate independently of or coincidental with the
host chromosomal DNA. Alternatively, the vector can target
insertion of the foreign or heterologous DNA into a host
chromosome.
[0028] The term "transgenic" refers to tobacco plant cells that
have been "transformed." "Transformed" describes the introduction
of DNA into the tobacco plant cell. In most cases the DNA is
introduced into the tobacco plant cell in the form of a vector
containing the DNA segment. A transformed tobacco plant may be
identified by selectable marker and report genes in accordance with
methods known in the art. "Expressed" describes a protein that is
produced in a plant cell when its DNA is transcribed to mRNA that
is translated to the protein. "Inhibition" describes a measurable
decrease in the cellular level of mRNA transcribed from the gene
(i.e., coding polynucleotide), and/or in the cellular level of a
peptide, polypeptide, or protein product of the coding
polynucleotide. "Overexpression" describes a greater expression
level of a gene in a tobacco plant or tobacco plant cell compared
to the expression in a wild-type tobacco plant or cell. "Suppressed
refers to decreased expression or activity of a protein.
[0029] The term "% sequence identity" describes the extent to which
the sequences of DNA or protein segments are invariant throughout a
window of alignment of sequences such as nucleotide sequences or
amino acid sequences and is determined by comparing two optimally
aligned sequences over a comparison window. An identity fraction
for a sequence aligned with a reference sequence is the number of
identical components which are shared by the sequences, divided by
the length of the alignment not including gaps introduced by the
alignment algorithm. "% identity" is the identity fraction times
100. "Substantially identical" describes nucleotide sequences that
are more than 85% identical to a reference sequence.
[0030] "Promoter" describes a regulatory DNA that initializes
transcription. Using methods known to a person of ordinary skill in
the art, recombinant DNA constructs are assembled and usually
include a promoter operably linked to DNA.
[0031] Definitions of common terms in cell biology and molecular
biology can be found in The Encyclopedia of Molecular Biology,
published by Blackwell Science Ltd., (1994) (ISBN 0-632-02182-9);
Benjamin Lewin, (2009) Genes X, published by Jones & Bartlett
Publishing, (ISBN-10: 0763766321); Kendrew et al. (eds.) (1995),
Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., (ISBN 1-56081-569-8)
and Current Protocols in Protein Sciences (2009), Wiley
Intersciences, Coligan et al., eds.
[0032] Unless otherwise stated, the present disclosure is performed
using standard procedures, as described, for example in Sambrook et
al., (2001) Molecular Cloning: A Laboratory Manual (3 ed.), Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA;
Davis et al., (1995) Basic Methods in Molecular Biology, Elsevier
Science Publishing, Inc., New York, USA; or Methods in Enzymology:
Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A.
R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); and
Current Protocols in Protein Science (CPPS) (John E. Coligan, et.
al., ed., John Wiley and Sons, Inc.), which are all incorporated by
reference herein in their entireties.
[0033] In some embodiments, tobacco plant genes encoding a
polypeptide, related to the biosynthesis of isomyosmine, may be
utilized to overexpress or inhibit the expression of the
polypeptide in the tobacco plant in which the polypeptide is
normally found. For example, the gene or a related vector may be
introduced into a cell of the plant in a genetic locus where the
gene is not normally found, or a native copy of the gene or related
vector may be placed under regulatory control elements that lead to
increased expression of the native gene or vector. In other
examples, the gene may be used to design a polynucleotide that
inhibits the expression of the gene, and the polynucleotide may be
introduced into a cell of the tobacco plant. Accordingly,
genetically modified tobacco plants may be engineered to increase
or decrease the levels of certain alkaloids, such as isomyosmine,
by overexpressing or inhibiting one or several key genes related to
alkaloid biosynthesis levels.
[0034] In one example, a tobacco may be made with increased levels
of isomyosmine by exposing a tobacco plant cell to a vector or
exogenous DNA construct that includes a promoter that is operable
in the tobacco plant cell and a DNA sequence capable of encoding an
enzyme(s) or protein(s) critical to the isomyosmine alkaloid
biosynthesis pathway inducing increased levels of isomyosmine
production and biosynthesis. Accordingly, the tobacco plant cell is
transformed by the DNA construct or vector. The transformed cells
are selected and the resulting transgenic tobacco plant is
regenerated using conventional techniques.
[0035] The expression of isolated nucleic acids encoding a protein
involved in the isomyosmine biosynthesis pathway can be achieved by
operably linking the DNA or cDNA to a promoter, and then
incorporated into an expression vector. Vectors can be suitable for
replication and integration in either prokaryotes or eukaryotes.
Typical expression vectors contain transcription and translation
terminators, initiation sequences, and promoters that regulate the
expression of the DNA encoding a protein involved in the
isomyosmine biosynthesis pathway. The vector is then introduced
into the appropriate host cell.
[0036] In accordance with one aspect of the current disclosure, a
genetically modified tobacco plant includes a tobacco plant
transformation vector comprising a nucleic acid encoding a
polypeptide having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%,
or 100% sequence identity to a tobacco plant gene related to
enzymes, proteins, or other components critical to the isomyosmine
alkaloid biosynthesis pathway, or directly related to the
biosynthesis and/or the metabolic pathway of isomyosmine. In
accordance with another aspect, the nucleic acid is operably linked
to a promoter. In accordance with another aspect, a nucleic acid
construct for genetically modified tobacco plants is provided. The
nucleic acid construct comprises a polynucleotide sequence encoding
a polypeptide having at least 80% sequence identity to a tobacco
plant gene related to the isomyosmine alkaloid biosynthesis
pathway, and one or more control sequences for driving expression
of the polynucleotide sequence in the genetically modified tobacco
plant.
[0037] Unless otherwise stated, "isomyosmine," as used herein,
refers to isomyosmine that has been prepared synthetically,
isomyosmine that has been produced from natural materials in which
it occurs, or isomyosmine that has been produced from a genetically
modified tobacco plant. The amount of isomyosmine in a solid
tobacco product composition usually ranges from about 0.001 mg/g to
about 10 mg/g, from about 0.01 mg/g to about 10 mg/g, or from about
0.1 mg/g to about 100 mg/g. Desirable isomyosmine content in a
tobacco product that is a solvent ranges from about 0.001 to about
10 mg/ml, often from about 0.01 to about 5 mg/ml, from about 0.05
to about 4 mg/ml, from about 0.1 to about 3 mg/ml, from about 0.2
to about 2.5 mg/ml, from about 0.3 to about 2 mg/ml, from about 0.2
to about 1.5 mg/ml, or from about 0.1 to about 1 mg/ml.
[0038] According to certain aspects, the transgenic tobacco plants,
and related tobacco and tobacco products contain reduced nicotine
levels that may reduce smoking behavior. Smoking behaviors may be
reduced further and more effectively with the incorporation of
increased isomyosmine levels as presently disclosed. Nicotine
dependence may be reduced in current smokers and tobacco users, new
users may be less likely to develop nicotine dependence and
continue to smoke, and former smokers and tobacco users who lapse
may be less likely to become regular users again. Unlike "light"
cigarettes, very low nicotine content cigarettes contain
substantially less nicotine in the tobacco. For example, the
tobacco described in the present disclosure includes nicotine
levels of less than 2 mg/g compared to 10-14 mg/g in a typical
cigarette. Typical nicotine levels in other tobacco products, such
as chewing tobacco or snuff, ranges from about 5-10 mg/g.
Henningfield, J. E., Radzius, A., & Cone, E. J. (1995),
Estimation of available nicotine content of six smokeless tobacco
products. Tobacco Control, 4 (1), 57-61. E-cigarettes, assuming a
series of 15 puffs is equivalent to smoking one cigarette, delivers
about 0.025-0.77 mg nicotine, compared to one smoked tobacco
cigarette of about 1.54-2.60 mg. Maciej L. Goniewicz, Ph.D., Tomasz
Kuma, M. Pharm., Michal Gawron, M. Pharm., Jakub Knysak, M. Pharm.,
Leon Kosmider, M. Pharm.; Nicotine Levels in Electronic Cigarettes,
Nicotine & Tobacco Research, Volume 15, Issue 1, 1 Jan. 2013,
Pages 158-166, https://doi.org/10.1093/ntr/nts103.
[0039] In some aspects, tobacco product of the disclosure may
include, but are not limited to, a cigarette, cigar, pipe tobacco,
smokeless tobacco, chewing tobacco, capsule, tablet, or lozenge.
According to other aspects, the tobacco products may be produced
from the genetically modified tobacco plant of the present
disclosure. According to other aspects, the modified tobacco of the
present disclosure is suitable for conventional growing and
harvesting techniques and the harvested tobacco leaves and stems
are suitable for use in one or more of the tobacco products
disclosed herein. In some aspects modified tobacco of the present
disclosure can be processed and blended with conventional
tobacco.
[0040] The description of embodiments of the disclosure is not
intended to be exhaustive or to limit the disclosure to the precise
form disclosed. While specific embodiments of, and examples for,
the disclosure are described herein for illustrative purposes,
various equivalent modifications are possible within the scope of
the disclosure, as those skilled in the relevant art will
recognize. For example, while method steps or functions are
presented in a given order, alternative embodiments may perform
functions in a different order, or functions may be performed
substantially concurrently. The teachings of the disclosure
provided herein can be applied to other procedures or methods as
appropriate. The various embodiments described herein can be
combined to provide further embodiments. Aspects of the disclosure
can be modified, if necessary, to employ the compositions,
functions and concepts of the above references and application to
provide yet further embodiments of the disclosure. Moreover, due to
biological functional equivalency considerations, some changes can
be made in protein structure without affecting the biological or
chemical action in kind or amount. These and other changes can be
made to the disclosure in light of the detailed description. All
such modifications are intended to be included within the scope of
the appended claims.
[0041] Specific elements of any of the foregoing embodiments can be
combined or substituted for elements in other embodiments.
Furthermore, while advantages associated with certain embodiments
of the disclosure have been described in the context of these
embodiments, other embodiments may also exhibit such advantages,
and not all embodiments need necessarily exhibit such advantages to
fall within the scope of the disclosure.
[0042] The following examples are set forth as being representative
of the present disclosure. These examples are not to be construed
as limiting the scope of the present disclosure as these and other
equivalent embodiments will be apparent in view of the present
disclosure, figures and accompanying claims.
* * * * *
References